Method to determine zygosity of the fad3 gene in canola using end-point taqman pcr

ABSTRACT

The subject disclosure relates in part to endpoint TaqMan® PCR assays for the detection and high throughput zygosity analysis of the fad-3c gene in canola. The subject disclosure further relates, in part, to the use of wild type DNA as a reference for use in determining zygosity. These and other related procedures can be used to uniquely identify the zygosity and variety of canola lines comprising the subject gene. The subject disclosure also provides related kits for determining zygosity from a sample of a canola plant or seed, for example.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/550,170, filed Oct. 21, 2011, which is herein incorporated byreference in its entirety.

BACKGROUND OF THE DISCLOSURE

The genus Brassica includes canola, one of the world's most importantoilseed crops, and an important oilseed crop grown in temperategeographies. Canola has been traditionally characterized as Brassicanapus L. (a species derived as a result of inter-specific crosses ofBrassica rapa and Brassica oleracea) in which erucic acid andglucosinolates have been eliminated or significantly reduced throughconventional breeding. The majority of canola oil is in the form ofvegetable oils produced for human consumption. There is also a growingmarket for the use of canola oil in industrial applications.

The genus Brassica is comprised of three diploid species each whichpossess a unique genome which is labeled as either the A genome, Bgenome, or C genome. Brassica rapa plants possess a diploid A genome.Brassica nigra plants possess a diploid B genome. Brassica oleracea,plants posses a diploid C genome. Hybrids of these species can beproduced via crossing between two of the diploid species. Canola is anamphidiploid species considered to have arisen from the hybridization ofBrassica oleracea, having a diploid C genome, and Brassica rapa, havinga diploid A genome. Cytogenetic investigation revealed the AA and CCgenomes show a degree of relatedness, being partially homologous to oneanother and thought to have been derived from a common ancestor genome(Prakash and Hinata, 1980). Although technically classified as diploids,the genomes of both progenitor species contain a high percentage ofregions duplicative of one another (Song et al, 1991). Genetic analysisrevealed that the AA genome of Brassica rapa contributed ten chromosomesto Brassica napus, while Brassica oleracea contributed nine chromosomesfrom its CC genome as the maternal donor (Song et al, 1992).

The quality of edible and industrial oil derived from a particularvariety of canola seed is determined by its constituent fatty acids, asthe type and amount of fatty acid unsaturation have implications forboth dietary and industrial applications. Conventional canola oilcontains about 60% oleic acid (C18:1), 20% linoleic acid (C18:2) and 10%linolenic acid (18:3). The levels of polyunsaturated linolenic acidtypical of conventional canola are undesirable as the oil is easilyoxidized, the rate of oxidation being affected by several factors,including the presence of oxygen, exposure to light and heat, and thepresence of native or added antioxidants and pro-oxidants in the oil.Oxidation causes off-flavors and rancidity of as a result of repeatedfrying (induced oxidation) or storage for a prolonged period(auto-oxidation). Oxidation may also alter the lubricative and viscousproperties of canola oil.

Canola oil profiles which exhibit reduced levels of polyunsaturatedfatty acids and increased levels of monounsaturated oleic acid relativeto conventional canola oil are associated with higher oxidativestability. The susceptibility of individual fatty acids to oxidation isdependent on their degree of unsaturation. Thus, the rate of oxidationof linolenic acid, which possesses three carbon-carbon double bonds, is25 times that of oleic acid, which has only one carbon-carbon doublebond, and 2 times that of linoleic acid, which has two carbon-carbondouble bonds. Linoleic and linolenic acids also have the most impact onflavor and odor because they readily form hydroperoxides. High oleic oil(.gtoreq.70% oleic) is less susceptible to oxidation during storage,frying and refining, and can be heated to a higher temperature withoutsmoking, making it more suitable as cooking oil.

The quality of canola oil is determined by its constituent fatty acidssuch as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid(C18:3). Most canola cultivars normally produce oil with about 55-65%oleic acid and 8-12% linolenic acid. High concentrations of linolenicacid lead to oil instability and off-type flavor, while high levels ofoleic acid increase oxidative stability and nutritional value of oil.Therefore, development of canola cultivars with increased oleic acid andreduced linolenic acid is highly desirable for canola oil quality.

Two loci were identified and their genomic location mapped from a canolacultivar which possesses increased oleic acid and reduced linolenic acidquantities. One locus has a major effect, and the second locus has aminor effect on production of increased oleic acid and reduced linoleicacid. The major locus for high oleic acid (C18:1) was determined to bethe fatty acid desaturase-2 (fad-2) gene and it is located on linkagegroup, N5. The second locus is located on linkage group NI. One majorQuantitative Trait Loci (QTL) for linolenic acid (C18:3) is the fattyacid desaturase-3 gene of the genome C (fad-3c) and it is located onlinkage group N14. The second major QTL resides on the N4 linkage groupand is the fatty acid desaturase-3 gene of the genome A (fad-3a).Genomic sequences of the fad-2 and fad-3c geneswere amplified andsequenced from both an ethly methanesulfonate (EMS)-induced mutant and awild-type canola cultivar. A comparison of the mutant and wild-typeallele sequences of the fad-2 and fad-3c genes revealed singlenucleotide polymorphisms (SNPs) in the genes from the EMS mutatedplants. Based on the sequence differences between the mutant andwild-type alleles, two SNP markers, corresponding to the fad-2 andfad-3c gene mutations, were developed. (Hu et al., 2006).

Current methods for producing F₁ hybrid Brassica seeds have limitationsin terms of cost and seed purity. Generally, these methods requirestable, sib-incompatible and self-incompatible, nearly homozygousparental breeding lines, which parental breeding lines are availableonly after repeated selfing to generate inbred lines. Furthermore,inbreeding to develop and maintain the parental lines is accomplished bylabor intensive techniques, such as bud pollination, since Brassicahybrid seed production systems based on self-incompatible traits mustutilize strongly self-incompatible plants. Environmental conditionsduring the breeding process, such as temperature and moisture, typicallyaffect plant lipid metabolism, thus also affecting the content level offatty acids (Harwood, 1999). Environmental variability therefore makesthe phenotypic selection of plants less reliable. Deng and Scarth (1998)found that increase in post-flowering temperature significantly reducedthe levels of C18:3 and increased C18:1. Similar results were reportedin other studies (Yermanos and Goodin, 1965; Canvin, 1965).

Breeding for low linolenic varieties is particularly challenging sinceC18:3 content is a multi-gene trait and inherited in a recessive mannerwith a relatively low heritability. Genetic analysis of a populationderived from the cross between “Stellar” (having a low C18:3 content(3%)) and “Drakkar” (having a “conventional” C18:3 level (9-10%))indicated that the low C18:3 trait was controlled by two major loci withadditive effects designated L1 and L2 (Jourdren et al., 1996b). Thesetwo major loci controlling C18:3 content were found to correspond to twofad-3 (fatty acid desaturase-3) genes; one located on the A genome(originating from Brassica rapa) and the other on the C genome(originating from Brassica olecera) (Jourdren et al., 1996; Barret etal., 1999).

Traits that are continuously varying due to genetic (additive,dominance, and epistatic) and environmental influences are commonlyreferred to as “quantitative traits.” Quantitative traits may bedistinguished from “qualitative” or “discrete” traits on the basis oftwo factors: environmental influences on gene expression that produce acontinuous distribution of phenotypes; and the complex segregationpattern produced by multigenic inheritance. The identification of one ormore regions of the genome linked to the expression of a quantitativetrait led to the discovery of Quantitative Trait Loci (“QTL”). Thormannet al. (1996) mapped two QTL that explained 60% of the variance for thelinolenic content, while Somers et al. (1998) identified three QTL thatcollectively explained 51% of the phenotypic variation of C18:3 content.A three-locus additive model was also reported by Chen and Beversdorf(1990). Rucker and Robbelen (1996) indicated that several minor genesare most likely involved in the desaturation step.

Heritability for C18:3 content was estimated to be 26-59% (Kondra andThomas, 1975) (where the variability of heritability is a function ofgenetics as opposed to environmental factors). Complexity of theinheritance of linolenic acid may be due to the fact that linolenic acidcan be synthesized either from the desaturation of C18:2 or theelongation of C16:3 (Thompson, 1983).

In contrast to linolenic acid, inheritance of oleic acid is lesscomplex, and the heritability of oleic acid is relatively high. It isreported that high oleic acid content is controlled by a major locuscalled fad-2 (fatty acid desaturase 2) gene which encodes the enzymeresponsible for the desaturation of oleic acid to linoleic acid (C18:2)(Tanhuanpaa et al., 1998; Schierholt et al., 2001). All of thefunctional gene copies of the fad-2 gene that have been reported andmapped to date are located on the A-genome-originated linkage group N5(Scheffler et al., 1997; Schierholt et al., 2000). Chen and Beversdorf(1990) reported that the accumulation of oleic acid was controlled by attwo segregation genetic systems, one acting on chain elongation and theother involving desaturation. Heritability for C18:1 content wasestimated to be 53% to 78% (Kondra and Thomas 1975) and 94% (Schierholtand Becker, 1999), respectively. Due to the higher heritability, theexpression of C18:1 content is environmentally less affected andrelatively stable (Schierholt and Becker, 1999).

In Nexera™ canola germplasm, 1 to 2 genes are found to control C18:1content and at least 3 genes are involved in C18:3 expression (Nexera™is a trademark of Dow AgroSciences, LLC). In segregating progenies, thedistribution of seed C18:3 content is continuous, thereby making itdifficult to identify genotypic classes with desirable C18:3 levels. Inaddition, there is a low correlation in fatty acid content betweengreenhouse (GH) and field grown plants, further making it challenging toreliably select GH plants with desirable levels of C18:3.

Various methods can be used to detect the presence of a specific gene ina sample of plant tissue. One example is the Pyrosequencing technique asdescribed by Winge (Innov. Pharma. Tech, 00:18-24, 2000). In this methodan oligonucleotide is designed that overlaps the inserted DNA sequenceand the genomic DNA adjacent thereto. thereto The oligonucleotide ishybridized to a single-stranded PCR product (an “amplicon”) from theregion of interest (i.e., one primer in the inserted sequence and one inthe flanking genomic sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal that is measured. A light signalindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single or multi-baseextension. (This technique is usually used for initial sequencing, notfor detection of a specific gene when it is known.)

Fluorescence Polarization is another method that can be used to detectan amplicon. Following this method, an oligonucleotide is designed tooverlap the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

Molecular Beacons have been described for use in sequence detection.Briefly, molecular beacons comprise a FRET (fluorescence resonanceenergy transfer) oligonucleotide probe which may be designed such thatthe FRET probe overlaps the flanking genomic and insert DNA junction.The unique structure of the FRET probe results in it containingsecondary structure that keeps the fluorescent and quenching moieties inclose proximity. The FRET probe and PCR primers (one primer in theinsert DNA sequence and one in the flanking genomic sequence) are cycledin the presence of a thermostable polymerase and dNTPs. Followingsuccessful PCR amplification, hybridization of the FRET probe to thetarget sequence results in the removal of the probe secondary structureand spatial separation of the fluorescent and quenching moieties. Afluorescent signal indicates the presence of the flankinggenomic/transgene insert sequence due to successful amplification andhybridization.

Hydrolysis probe assays, also known as TaqMan® PCR (TaqMan® is aregistered trademark of Roche Molecular Systems, Inc.), provide a methodof detecting and quantifying the presence of a DNA sequence. Briefly,TaqMan® PCR utilizes a FRET oligonucleotide probe which is designed tohave a portion of the oligo within the transgene and another portion ofthe oligo within the flanking genomic sequence for event-specificdetection. The FRET probe and PCR primers (one primer in the insert DNAsequence and one in the flanking genomic sequence) are cycled in thepresence of a thermostable polymerase and dNTPs. Hybridization of theFRET probe, and subsequent digestion during the PCR amplification stagedue to 5′ exonuclease activity of the Taq polymerase, results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful hybridizationand amplification.

Molecular markers are also useful for sequence specific identificationof DNA. Molecular marker selection is based on genotypes and istherefore independent from environment effects. Molecular markers helpto alleviate the problem of the unreliable selection of plants in thegreenhouse attributable to the low correlation in fatty acid contentbetween greenhouse grown plants and field grown plants. Significantly,molecular markers tightly linked to the genes controlling C18:1 andC18:3 content can facilitate early selection of plants carrying genesfor high C18:1 and low C18:3. Marker-assisted selection at early stagecan help to save greenhouse space, improve the efficiency of greenhouseuse, and reduce breeding workload in the field.

More generally, molecular markers have advantages over morphologicalmarkers in that: molecular markers can be highly polymorphic whilemorphological markers are strictly phenotype dependent; morphologicalmarkers may interfere in the scoring of certain quantitative phenotypeswhile molecular markers exhibit a 1:1 relationship between genotype andphenotype (thus allowing the unambiguous scoring of all possiblegenotypes for a given locus); and epistatic interactions tend to limitthe number of morphological markers useful in a population, whilemolecular markers do not interact epistatically.

Different types of molecular markers such as RAPD (random-amplifiedpolymorphic DNA) markers (Tanhuanpaa et al., 1995; Hu et al., 1995;Rajcan et al., 1999; Jourdren et al., 1996), RFLP (restriction fragmentlength polymorphism) markers (Thormann et al., 1996) and SCAR(sequence-characterized amplified region) markers (Hu et al, 1999) havebeen identified to be associated with low C18:3 levels in Brassicanapus. Molecular markers have also been identified for high C18:1content. A RAPD marker was identified to be linked to the QTL affectingoleic acid concentration in spring turnip rape (B. rapa ssp. oleifera)and was later converted into a SCAR marker (Tanhuanpaa et al., 1996).Schierholt et al. (2000) identified three AFLP (amplified fragmentlength polymorphism) markers linked to a high oleic acid mutation inwinter oilseed rape (B. napus L.). Tanhuanpaa et al. (1998) developed anallele-specific PCR marker for oleic acid by comparing the wild-type andhigh-oleic allele of the fad-2 gene locus in spring turnip rape (B. rapassp. oleifera). However, most of these markers are low-throughputmarkers such as RAPD, AFLP and RFLP and are not suitable for large scalescreening through automation.

BRIEF SUMMARY OF THE DISCLOSURE

The subject disclosure relates in part to endpoint TaqMan® PCR assaysfor the detection, and high throughput zygosity analysis, of the fad-3cgene in canola. The subject disclosure further relates, in part, to theuse of wild-type fad-3c gene in canola as a reference for use indetermining zygosity. These and other related procedures can be used touniquely identify the zygosity and variety of canola lines comprisingthe subject gene.

The subject disclosure also provides related kits for determining thezygosity and variety from a sample (of canola, for example).

Thus, an embodiment of the subject disclosure relates to TaqMan® PCR, aflexible platform for high throughput zygosity and breeding analysis.Utilization of the end-point TaqMan® PCR application presented herewiththis disclosure provides a reliable, accurate, and high throughputapplication for fad-3c zygosity and breeding analysis of canola.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. is a section of fad-3c gene sequence (SEQ ID NO:1) illustratingthe position of the fad-3c mutation identified by Hu et al. (2006)(arrow). Intron 6 is in lighter colored text and a second polymorphismis indicated with an asterisk.

FIG. 2. is an example of zygosity analysis results (of canola), showingthree fad-3c genotypes following an end point TaqMan® assay (resultsgenerated using SDS 2.4 software available through Applied Biosystems,Foster City, Calif., USA).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 provides a section of the fad-3c gene sequence illustratingthe position of the fad-3c mutation.

SEQ ID NO:2 provides forward primer D-CL-FAD3C-F (which binds flankinggenomic sequence).

SEQ ID NO:3 provides reverse primer D-CL-FAD3C-R2 (which binds insertionsequence).

SEQ ID NO:4 provides probe D-CL-FAD3C-FAM for preferential binding ofmutated fad-3c gene having a G to A single nucleotide polymorphism.

SEQ ID NO:5 provides probe D-CL-FAD3C-VIC for detection of wild typefad-3c gene.

DETAILED DESCRIPTION OF THE DISCLOSURE

The subject disclosure relates in part to endpoint TaqMan® PCR assaysfor the detection and high throughput zygosity analysis of the fad-3cgene in canola. The subject disclosure further relates, in part, to theuse of wild-type fad-3c gene in canola as a reference for use indetermining zygosity. These and other related procedures can be used touniquely identify the zygosity and variety of canola lines comprisingthe subject gene. The subject disclosure also provides related kits fordetermining the zygosity and variety from a sample (of canola, forexample). Thus, an embodiment of the subject disclosure relates toTaqMan® PCR, a flexible platform for high throughput zygosity andbreeding analysis. Utilization of the end-point TaqMan® PCR applicationpresented herewith this disclosure provides a reliable, accurate, andhigh throughput application for fad-3c zygosity and breeding analysis ofcanola.

Novel assays of the subject invention were developed based in part on asingle nucleotide polymorphism (SNP) mutation of the fad-3c allelereported by Hu et al. (2006). The assay utilizes two primer regions andtwo MGB probes to detect mutant and wild type fad-3c alleles (see Table1). TaqMan® primers and probes to detect this SNP mutation were designedin part by Primer express software (Applied Biosystems, Austin, Tx)using the fad-3c gene sequences. This new fad-3c TaqMan® assay wasvalidated using DNA extracted from canola plants which are homozygous,hemizygous and wild type (no mutation) for the fad-3c gene. The fad-3cTaqMan® assay was also optimized for performance in part with theApplied Biosystems 7900HT Real-Time PCR system on both the 96 or 384well formats using fast PCR thermal cycling conditions

TABLE 1 Primer and probe sequences used in the fad-3c TaqMan ® assaySEQ ID Descrip- NO: Name tion Sequence (5′-3′) SEQ ID D-CL- ForwardACGATGATAAGCTGCCTTGGT NO: 2 FAD3c-F primer SEQ ID D-CL- ReverseTCAACAGTTGTTAATCCTCCACGT NO: 3 FAD3c-R primer SEQ ID D-CL- Probe to6FAM-CAGAGGCAAGATAAGT-MGB NO: 4 FAD3c- detect FAM fad-3c mutant SEQ IDD-CL- Probe to VIC-ACAGAGGCAAGGTAAGT-MGB NO: 5 FAD3c- detect VIC fad-3cwild type

NEX828 and Quantum leaf samples were used in the assay. DNA from canolabreeding populations were used to validate this assay.

Aspects of the subject disclosure include methods of designing and/orproducing diagnostic nucleic acid molecules exemplified and/or suggestedherein. Specific TaqMan® primers and probe were designed, as detailedherein, in part according to the DNA sequences located at, or inproximity upstream or downstream to, the specific SNPs identified hereinin the fad-3c gene.

Thus, in some embodiments, this disclosure relates to determiningzygosity of canola oil producing plants. The subject disclosure relatesin part to detecting the presence of SNPs identified herein, in order todetermine whether progeny of a sexual cross contain the SNPs ofinterest, and the zygosity of the progeny. In addition, methods fordetecting zygosity are included and are helpful, for example, forcomplying with regulations requiring the pre-market approval andlabeling of foods derived from recombinant crop plants.

The subject disclosure relates in part to a fluorescence-based endpointTaqMan® PCR assay utilizing the endogenous, non-mutant fad-3c gene as acontrol for high-throughput zygosity analysis of canola plants.

The subject disclosure also relates in part to the development of abiplex endpoint TaqMan® PCR for canola zygosity analysis. Further, thesubject disclosure relates in part to the development of canola fad-3cgene breeding test kits.

In general, endpoint TaqMan® assays are based on a plus/minus strategy,by which a “plus” signifies the sample is positive for the assayed geneand a “minus” signifies the sample is negative for the assayed gene.These assays typically utilize one set of oligonucleotide primers andtwo oligonucleotide probes, one probe preferentially hybridizing themutated fad-3c SNP and the other probe preferentially hybridizing thewild-type fad-3c sequence, respectively.

Advantages associated with the subject disclosure include its decreasedreliance on DNA quality and quantity. Further, the subject disclosuredoes not require a lengthy initial denaturing step which, if not handledproperly, can often render other SNP detection assays unsuccessful.Additionally, the subject disclosure is provides a method to efficientlyanalyze large numbers of canola samples in a high-throughput mannerwithin a commercial setting. Another advantage of the subject disclosureis time savings. The subject Endpoint TaqMan® analysis for canolazygosity and breeding analysis offers advantages over other applicationformats, particularly when analyzing large numbers of samples.

This disclosure relates in part to plant breeding analysis. Thisdisclosure includes novel detection of methods for SNPs in canola plantsthat affect oleic and linolenic acid levels in the subject plants.

Further, it may be possible to detect the presence of the subject SNPsby other known nucleic acid detection methods, such as PCR or DNAhybridization using the nucleic acid probes described herein.Event-specific PCR assays are discussed herein. (See also Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459462, 1999.)

As used herein, the term “progeny” denotes the offspring of anygeneration of a parent plant.

Detection techniques of the subject disclosure are especially useful inconjunction with plant breeding, for example, to determine zygosity ofprogeny plants after a parent plant comprising a SNP of interest iscrossed with another plant. The subject application and methods benefitcanola breeding programs as well as quality control processes. PCRdetection kits for canola lines, using the methods and assays disclosedherein can now be made and used. Further, the subject disclosure maybenefit product registration and product stewardship.

A canola plant comprising desired fad-3c genetic composition can be bredby first sexually crossing a first parental canola plant consisting of acanola plant grown from seed of any one of the lines referred to herein,and a second parental canola plant, thereby producing a plurality offirst progeny plants; and then selecting a first progeny plantpossessing desired fad-3c genes as disclosed by the subject disclosure;and selfing the first progeny plant, thereby producing a plurality ofsecond progeny plants; and then selecting from the second progeny plantsa plant that possesses desired fad-3c genes according to the subjectdisclosure. These steps can further include the back-crossing of thefirst progeny plant or the second progeny plant to the second parentalcanola plant or a third parental canola plant. A canola crop comprisingcanola seeds of the subject disclosure, or progeny thereof, can then beplanted.

This disclosure further includes processes of making crosses usingcanola plant comprising the desired fad-3c genetic composition as atleast one parent. For example, the subject disclosure includes an F₁hybrid plant having as one or both parents any of the canola plantcomprising the desired fad-3c genetic composition. Also within thesubject disclosure is seed produced by such F₁ hybrids. This disclosureincludes a method for identifying an F₁ hybrid seed by crossing anexemplified plant with a different (e.g. in-bred parent) plant andharvesting and assaying the resultant hybrid seed, using the method ofthe subject disclosure. The canola plants that are used to produce theF₁ hybrid may be either a female parent or a male parent.

It is also to be understood that transgenic plants may be produced tocontain the fad-3c genes disclosed herein. Additionally, transgenicplants comprising the fad-3c gene characteristics disclosed herein maybe mated with a plant comprising a different genetic composition,thereby producing offspring containing independently segregatingexogenous genes. Selfing of appropriate progeny can produce plants thatare homozygous for the added, exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Other breeding methodscommonly used for different traits and crops are known in the art.Backcross breeding has been used to transfer genes for a simplyintrogressed, highly heritable trait into a desirable homozygouscultivar or inbred line, which is the recurrent parent. The source ofthe trait to be transferred is called the donor parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.After the initial cross, individuals possessing the phenotype of thedonor parent are selected and repeatedly crossed (backcrossed) to therecurrent parent. The resulting parent is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent. The method of the subjectdisclosure provides a high-throughput, fluorescence-based endpointTaqMan® PCR assay to detect the fad-3c transgene in progeny plants andto determine the zygosity level of progeny plants.

The methods of the present disclosure, for example the oligonucleotideprimers and probes, can be used for marker assisted breeding (MAB)methods. The methods of the present disclosure, for example theoligonucleotide primers and probes, can be used with related assaysAmplified Fragment Length Polymorphism assays (AFLP) RestrictiveFragment Length Polymorphism assays (RFLP) Random Amplified PolymorphismDNA assays (RAPD)) that identify genetically linked agronomically usefultraits by the detection of SNPs or Simple Sequence Repeats (SSRs), usingpublicly available protocols that are known in the art. The SNPsdisclosed herein can be tracked in the progeny of a cross with a canolaplant of the subject disclosure (or progeny thereof and any other canolacultivar or variety) using the MAB methods. DNA molecules can be used asmarkers for this trait, and MAB methods that are well known in the artcan be used to track the SNPs in canola plants where at least one canolaplant of the subject disclosure, or progeny thereof, was a parent orancestor. The methods of the present disclosure can be used to identifyany canola variety having the subject SNPs disclosed herein.

Methods of the subject disclosure include a method of producing a canolaplant comprising a combination of the SNPs identified herein, whereinsaid method comprises breeding with a plant of the subject disclosure.More specifically, said methods can comprise crossing two plants of thesubject disclosure, or one plant of the subject disclosure and any otherplant. Exemplary methods may further comprise selecting progeny of saidcross by analyzing said progeny for a SNP of the subject disclosure,detectable according to the subject disclosure. For example, the subjectdisclosure can be used to track the zygosity of canola plants throughbreeding cycles with plants comprising other desirable traits, such asagronomic traits such as those tested herein in various Examples. Plantscomprising the subject SNPs and the desired traits may also be detected,identified, selected, and quickly used in further rounds of breeding,for example. The subject SNPs/traits can also be combined throughbreeding, and tracked according to the subject disclosure, with othertraits, for example possible insect resistant trait(s) and/or herbicidetolerance traits. One embodiment of the latter is a plant comprising oneor more of the subject SNPs combined with a gene encoding resistance toa herbicide such as glyphosate.

In some embodiments, the present disclosure includes DNA sequences thatcomprise a contiguous fragment useful as primer sequences for theproduction of an amplicon product diagnostic for one or more of thefad-3c canola plants.

Related embodiments pertain to DNA sequences that comprise at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or more contiguous nucleotides of a portion of DNA sequencesidentified herein, or complements thereof. Such sequences may be usefulas DNA primers in DNA amplification methods. The amplicons producedusing these primers may be diagnostic for any combination and zygosityof fad-3c canola varieties referred to herein. Therefore, the disclosurealso includes the amplicons produced by such DNA primers and homologousprimers.

In still further embodiments, the subject disclosure includes methods ofproducing fad-3c SNPs of the subject disclosure, wherein said methodcomprises the steps of: (a) sexually crossing a first parental canolaline comprising one of the SNPs disclosed herein and conferring one ofthe oleic and/or linolenic acid traits disclosed here) and a secondparental canola line (that lacks these SNPs) thereby producing aplurality of progeny plants; and (b) selecting a progeny plant by theuse of molecular markers. Such methods may optionally comprise thefurther step of back-crossing the progeny plant to the second parentalcanola line to produce a true-breeding or homozygous canola plant thatcomprises said fad-3c traits.

According to another aspect of the disclosure, methods of determiningthe zygosity of progeny of a cross with said fad-3c canola plants isprovided. Said methods can comprise contacting a sample, comprisingcanola DNA, with a primer set of the subject disclosure. Said primers,when used in a nucleic-acid amplification reaction with genomic DNA fromat least one of said fad-3c canola plants, produces a first ampliconthat is diagnostic for at least one of said canola SNPs or wild typegenes. Such methods further comprise performing a nucleic acidamplification reaction, thereby producing the first amplicon anddetecting the first amplicon with probes specific for the SNPs of thefad-3c disclosed herein and the wild type genes. The methods furthercomprise performing allelic discrimination melting applications of theamplicons having the disclosed probes annealed thereto, and comparingthe relative florescence of the probes used in the allelicdiscrimination melting application. The relative florescence of theprobes indicates whether the sample contains the SNP of interest, and ifso, whether the sample is heterozygous or homozygous for the SNP.

DNA detection kits can be developed using the compositions disclosedherein, in conjunction with methods well known in the art of DNAdetection. The kits are useful for identification of the subject canolaSNPs in a sample and can be applied to methods for breeding canolaplants containing this DNA. The kits contain DNA sequences homologous orcomplementary to the amplicons, for example, disclosed herein. These DNAsequences can be used in DNA amplification reactions or as probes in aDNA hybridization method. The kits may also contain the reagents andmaterials necessary for the performance of the detection method.

A “probe” is an isolated nucleic acid molecule to which is attached aconventional detectable label or reporter molecule (such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme). Such aprobe is complementary to a strand of a target nucleic acid, in the caseof the present disclosure, to a strand of genomic DNA from one of saidcanola plants comprising fad-3c genes of interest, whether from a canolaplant or from a sample that includes DNA from the event. Probesaccording to the present disclosure include not only deoxyribonucleic orribonucleic acids but also polyamides and other probe materials thatbind specifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

Specific probes were designed comprising a fluorescent reporter(fluorophore) and a quencher that hybridizes to the target DNA betweenthe PCR primers. The fluorophore molecule is added to an oligonucleotideprobe during the synthesis of the oligonucleotide probe thereby labelingthe oligonucleotide probe. Other molecules can be added tooligonucleotide probe, such as a quencher molecule. The addition ofthese molecules to an oligonucleotide probe does not impair the functionof the oligonucleotide probe when hybridizing to single stranded DNA andproducing a new strand of DNA via an amplification process.

Numerous fluorophores have been developed that excite at specificwavelengths and are known in the art. Excitation of the fluorophoreresults in the release of a fluorescent signal by the fluorophore whichcan be quenched by a quencher located in close proximity to thefluorophore. When the quencher is disassociated from the fluorophore,the fluorescent signal is no longer quenched and accumulation of thefluorescent signal, which is directly correlated with the amount oftarget DNA, can be detected in real-time with an automated fluorometer.The fluorophores may be used in combination, wherein the excitation andemission spectra are significantly differ as to allow multiple detectionof two or more fluorophores. Some preferred embodiments of fluorophoresinclude; a HEX fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, or a ROX fluorescent dye.One preferred embodiment of a fluorophore for use with the methodconsisting of a homogeneous assay detection system for a PCR processusing FRET of the subject invention includes a FAM fluorescent dye of aJOE fluorescent dye.

Quenchers have been developed to quench fluorophores at a specificwavelength and are known in the art. When the quencher is located inclose approximation to the fluorophore, the fluorophore transfers energyto the quencher. The quencher transfer this energy and returns to anative ground state through emissive decay or nonradiatively. Innonradiative or dark decay, the energy transferred from the fluorophoreis given off as molecular vibrations. Selection of a quencher considersqualities such as low background fluorescence, high sensitivity, andmaximal spectral overlap to provide a quencher that can enable a wideruse of fluorophores. Some preferred embodiments of quenchers include;Dabcyl quenchers, Tamra quenchers, Qxl quencher, Iowa black FQ quencher,Iowa black RQ quencher, or an IR Dye QC-1 quencher. An especiallypreferred embodiment of a quencher would include an Blackhole quencherlabeled on an oligonucleotide primer which is designed antisense to theFAM labeled oligonucleotide.

“Primers” are isolated/synthesized nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization, therebyforming a hybrid between the primer and the target DNA strand and thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs of the present disclosure refer to their usefor amplification of a target nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other conventional nucleic-acidamplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, or 500 polynucleotides or more in length. Such probes andprimers hybridize specifically to a target sequence under highstringency hybridization conditions. Preferably, probes and primersaccording to the present disclosure have complete sequence similaritywith the target sequence, although probes differing from the targetsequence and that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989. PCR-primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose.

Primers and probes based on the DNA sequences upstream and downstream ofthe SNPs disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed sequences by conventional methods, e.g., byre-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present disclosure hybridizeunder stringent conditions to a target DNA sequence. In general, anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a fad-3c sample. Nucleic acidmolecules or fragments thereof are capable of specifically hybridizingto other nucleic acid molecules under certain circumstances. As usedherein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989. Departures fromcomplete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure. In order for a nucleic acid moleculeto serve as a primer or probe it need only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52and 9.56-9.58. Accordingly, the nucleotide sequences of the disclosuremay be used for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments.

Depending on the application envisioned, one can use varying conditionsof hybridization to achieve varying degrees of selectivity of probetowards target sequence. For applications requiring high selectivity,one will typically employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.50 mM to about 02.00mM MgCl₂ at temperatures of about 50° C. to about 75° C. Bothtemperature and salt may be varied, or either the temperature or thesalt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses.

In one exemplary embodiment, a nucleic acid of the present disclosurewill specifically hybridize to one or more of the primers (or ampliconsor other sequences) exemplified or suggested herein, includingcomplements and fragments thereof, under high stringency conditions. Inone aspect of the present disclosure, a marker nucleic acid molecule ofthe present disclosure has the nucleic acid sequence as set forth hereinin one of the exemplified sequences, or complements and/or fragmentsthereof.

In another aspect of the present disclosure, a marker nucleic acidmolecule of the present disclosure shares between 80% and 100% or 90%and 100% sequence identity with such nucleic acid sequences. In afurther aspect of the present disclosure, a marker nucleic acid moleculeof the present disclosure shares between 95%, 96%, 97%, 98%, and/or 99%and 100% sequence identity with such sequence. Such sequences may beused as markers in plant breeding methods to identify the progeny ofgenetic crosses. The hybridization of the probe to the target DNAmolecule can be detected by any number of methods known to those skilledin the art, these can include, but are not limited to, fluorescent tags,radioactive tags, antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridizeprimarily to, and with a high preference for, their target nucleic-acidsequences, thereby allowing the primer pair to bind and, preferably,produce a unique amplicon.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes, under stringent hybridization conditions, primarilyto, and with a high preference for, the nucleic acid sequence in asample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thecanola plant resulting from a sexual cross contains a SNP of interest asdisclosed herein. DNA extracted from a canola plant tissue sample may besubjected to a nucleic acid amplification method using a primer pairthat includes a primer derived from an upstream or downstream sequencein the genome of the canola plant adjacent to the SNP site and a secondprimer derived from the other end of the upstream or downstream sequencein the genome of the canola plant adjacent to the SNP site therebyproducing an amplicon that is diagnostic for the presence of the SNP.The amplicon is of a length and has a sequence that is also diagnosticfor the wild type or mutatedgene. The amplicon may range in length fromthe combined length of the primer pairs plus one nucleotide base pair,and/or the combined length of the primer pairs plus about 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250,1500, 1750, 2000, or more nucleotide base pairs (plus or minus any ofthe increments listed above). A member of a primer pair derived from theplant genomic sequence may be located a distance from the SNP sequence.This distance can range from one nucleotide base pair up to about twentythousand nucleotide base pairs. The use of the term “amplicon”specifically excludes primer dimers that may be formed in the DNAthermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including PCR. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR amplificationmethods have been developed to amplify up to 22 kb of genomic DNA. Thesemethods as well as other methods known in the art of DNA amplificationmay be used in the practice of the present disclosure. The sequence of afad-3c SNP can be verified by amplifying such sequences using primersderived from the sequences provided herein followed by standard DNAsequencing of the PCR amplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. Agarose gel electrophoresis and staining with ethidiumbromide is a common well known method of detecting DNA amplicons.Another such method is Genetic Bit Analysis where a DNA oligonucleotideis designed which overlaps both the adjacent flanking genomic DNAsequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking genomic sequence), a single-stranded PCR product canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

TaqMan® PCR is a method of detecting and quantifying the presence of aDNA sequence. Briefly, a FRET oligonucleotide probe is designed that itoverlaps a SNP of interest. The FRET probe and PCR primers (at least oneupstream and at least one downstream of the SNP of interest) are cycledin the presence of a thermostable polymerase and dNTPs.

Following amplification, allelic discrimination analysis (using theTaqMan® hydrolysis probe described above), may be performed fordetermining the presence of a SNP of interest and the zygosity of thesample. During allelic discrimination analysis, two differenthybridization probes (one probe including a nucleotide complementary tothe SNP sequence and the other probe having a nucleotide complementaryto the wild type sequence) are hybridized to the amplicon and digested,thereby releasing the quencher moieties from the probe due to the 5′exonuclease activity of the taq polymerase and resulting influorescence. A comparison of the relative fluorescence of a probespecific for the wild type gene versus a probe specific for the SNPprovides an indication of the presence and zygosity of the SNP ofinterest.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

The following examples are included to illustrate procedures forpracticing the disclosure and to demonstrate certain preferredembodiments of the disclosure. These examples should not be construed aslimiting. It should be appreciated by those of skill in the art that thetechniques disclosed in the following examples represent specificapproaches used to illustrate preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in these specific embodimentswhile still obtaining like or similar results without departing from thespirit and scope of the disclosure. Unless otherwise indicated, allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

The following abbreviations are used unless otherwise indicated.

-   -   bp base pair    -   ° C. degrees Celcius    -   DNA deoxyribonucleic acid    -   FRET fluorescence resonance energy treansfer    -   DIG digoxigenin    -   EDTA ethylenediaminetetraacetic acid    -   kb kilobase    -   μg microgram    -   μL microliter    -   mL milliliter    -   M molar mass    -   OLP overlapping probe    -   PCR polymerase chain reaction    -   PTU plant transcription unit    -   SDS sodium dodecyl sulfate    -   SNP single nucleotide polymorphism    -   SOP standard operating procedure    -   SSC a buffer solution containing a mixture of sodium chloride        and sodium citrate, pH 7.0    -   TBE a buffer solution containing a mixture of Tris base, boric        acid and EDTA, pH 8.3    -   V volts

EXAMPLES Example 1: FAD-3c End Point TAQMAN® Assay

An end-point TaqMan® assay was developed to detect the fad-3c singlenucleotide polymorphism mutation and to determine zygosity status ofcanola plants containing the fad-3c gene mutation in breedingpopulations. Two primers were designed to bind highly conserved DNAsequences located on exon 6 and 7 of the fad-3c gene. These primersamplified a 154 bpDNA fragment which spanned across the fad-s3c singlenucleotide polymorphism in mutated and un-mutated canola plants. Thefad-3c mutation in canola is described by Hu et al. (2006) and locatedin the exon 6 and intron 6 splice site junction of the gene (FIG. 1).Two TaqMan® minor groove binding non-fluorescent quencher (MGBNFQ)probes were designed with FAM and VIC as reporter dyes to detect thepresence of the wild type fad-3c gene and the mutated fad-3c gene (whichconsists of a single nucleotide polymorphism, SNP), respectively. Thesetwo probes were designed with special considerations to avoid aneighboring polymorphism located on the intron 6 and in close proximityto the fad-3c single nucleotide polymorphism (FIG. 1). Avoiding thesecond polymorphism resulted in increased specificity of the probes fordetection of the fad-3c mutant plants. The TaqMan® detection method forcanola plants containing the fad-3c SNP was tested against canolavariety “NEX 828” (containing the fad-3c SNP), control canola variety“Quantum” (does not contain the fad-3c SNP) and a DNA sample isolatedfrom plants known to be heterozygous for the fad-3c SNP. The end-pointTaqMan® assay was used to determine the presence of the fad-3c SNP andalso to determine the zygosity of the plants being tested in a highthroughput application, for example 96 and 384 well plate formats.

Example 1.1: gDNA Isolation

Genomic DNA (gDNA) samples of 156 different canola plants containing thefad-3c mutation and control canola plants were tested in this study.Genomic DNA was extracted using modified Qiagen MagAttract plant DNA kit(Qiagen, Valencia, Calif.). Fresh canola leaf discs, 4 per sample, wereused for gDNA extraction. The gDNA was quantified with the Pico Greenmethod according to vendor's instructions (Molecular Probes, Eugene,Oreg.). Samples were diluted with DNase-free water resulting in aconcentration of 5 ng/μL for the purpose of this study.

Example 1.2: TaqMan® Assay and Results

Specific TaqMan® primers and probes were designed for use in a TaqMan®end point assay. These primers and probes were designed to amplify anddetect the region of the fad-3c gene comprising the SNP of interest.These reagents can be used with the conditions listed below to detectthe mutated fad-3c gene within canola plants. Table 1 lists the primerand probe sequences that were developed specifically for the detectionof the fad-3c SNP in canola plants.

TABLE 1 Tagman PCR Primers and Probes SEQ ID Descrip- NO: Name tionSequence SEQ ID D-CL- Forward ACGATGATAAGCTGCCTTGGT NO: 2 FAD3c- primerF SEQ ID D-CL- Reverse CAAGTACCTCAACAACCCTTTGGTC NO: 3 FAD3c- primerAACAGTTGTTAATCCTCCACGT R SEQ ID D-CL- Probe to 6FAM-CAGAGGCAAGATAAGT-MGBNO: 4 FAD3c- detect FAM fad-3c mutant SEQ ID D-CL- Probe toVIC-ACAGAGGCAAGGTAAGT-MGB NO: 5 FAD3c- detect VIC fad-3c wild type

The PCR reaction mixtures for amplification are as follows: 1× TaqMan®GTExpress Master Mix, 0.9 μM forward primer (SEQ ID NO:2), 0.9 μMreverse primer (SEQ ID NO:3), 0.2 μM FAD-3C mutant probe (SEQ ID NO:4),0.2 μM wild type Probe (SEQ ID NO:5), 15 ng gDNA in a total reaction of6 μl. The reaction mixture was amplified using the following thermalcycling conditions: initial two steps of 50° C. for 2 min and 95° C. for30 sec; followed by 40 cycles of 3 seconds at 95° C. and 30 seconds at62° C. The reactions were then kept at 10° C. until being removed fromthe thermal cycler. PCR thermal cycling can be performed ether usingABI-Applied Biosystems 7900 HT real time PCR system or AppliedBiosystems Verity thermal Cyclers (Life Technologies, Carlsbad, Calif.).The sample plates consisted of control DNA from canola plants that werehomozygous for the fad-3c mutant (“NEX 828”), heterozygous for thefad-3c mutant, or homozygous for the fad-3c wild type (“Quantum”). Inaddition, a no template control which did not contain DNA was included.After amplification the end point florescent signals (VIC and FAM) wereread using Applied Biosystems 7900 HT real time PCR system according tothe allelic discrimination plate reading procedure as described by themanufacturer. The data was then analyzed using SDS 2.4 software (LifeTechnologies, Carlsbad, Calif.) to determine the relative fluorescenceof each sample (FIG. 2).

The TaqMan® detection method for the fac-3c mutation in canola wastested against known homozygous, hemizygous, and wildtype samples. Ananalysis of the florescence produced from each probe (of a sample'sreaction), with the florescence produced by the probes of the controls,aides in determining the zygosity of each sample. This assaydemonstrated high specificity for the detection of the fad-3c mutationand wildtype single nucleotide polymorphism in canola and did notproduce or amplify any detectable false-positive results from thecontrols. The event specific primers and probes can be used for thedetection of the fad-3c mutant and fad-3c wildtype gene in canola andthese conditions and reagents are applicable for zygosity assays.

1-16. (canceled)
 17. A method for determining zygosity of a canola plantcomprising a fad-3c gene, said method comprising: obtaining a sample ofgenomic DNA from a canola plant; hybridizing the sample of genomic DNAwith a first primer, a second primer, a first probe, and a second probe,wherein said first primer and said second primer comprise SEQ ID NO: 2and SEQ ID NO: 3, and said first probe and said second probe compriseSEQ ID NO: 5 and SEQ ID NO: 4, wherein each of said first probe and saidsecond probe is labeled with a fluorescent dye and a quencher; measuringflorescence of said first probe, said second probe, or a combinationthereof; and determining zygosity of said canola plant.
 18. The methodof claim 17, wherein the sample of genomic DNA comprises a mutatedfad-3c sequence having a single nucleotide polymorphism, wherein saidsingle nucleotide polymorphism consists of a G-to-A polymorphism. 19.The method of claim 18, wherein the sample of genomic DNA furthercomprises a wild-type fad-3c sequence.
 20. The method of claim 17,wherein said method is used for breeding introgression verification ofcross-bred canola plants.
 21. The method of claim 17, wherein said firstprobe comprises VIC as said fluorescent dye at the 5′ end of said firstprobe and a MGB quencher on the 3′ end of said first probe.
 22. Themethod of claim 17, wherein said second probe is labeled with FAM at the5′ end of said second probe and a MGB quencher at the 3′ end of saidsecond probe.
 23. The method of claim 17, wherein measuring florescencecomprises measuring and analyzing fluorescence directly in a platereader.
 24. The method of claim 17, wherein said DNA sample is obtainedfrom a canola plant in a field.
 25. The method of claim 17, wherein saidfirst probe and said second probe hybridizes to the sample of genomicDNA for a period of time and at a temperature of 50-70 degrees Celsius.26. The method of claim 25, further comprising increasing saidtemperature after the period of time.
 27. The method of claim 26,wherein said florescence produced by each of said first probe and saidsecond probe during the step of increasing said temperature is measuredby increments.
 28. The method of claim 17, wherein said first probehybridizes to a region of a wild-type fad-3c sequence and said secondprobe hybridizes to a region of a mutated fad-3c sequence having asingle nucleotide polymorphism (SNP).
 29. A kit for performing themethod of claim 17, said kit comprising said first primer, said secondprimer, said first probe, and said second probe.
 30. The kit of claim29, wherein said first primer is SEQ ID NO:2, said second primer is SEQID NO:3, said first probe is SEQ ID NO:5, and said second probe is SEQID NO:4.